Grewiifopenes A–K, bioactive clerodane diterpenoids from Casearia grewiifolia Vent.

Eleven novel clerodane-type diterpenoids, grewiifopenes A–K (1–4 and 12–18), along with nine known compounds (5–11, 19, and 20) were purified from the dichloromethane extract of the twigs and stems of Casearia grewiifolia Vent. (Salicaceae). Their spectroscopic data, including the NMR, HRESIMS, and electronic circular dichroism calculations were employed to completely characterize and elucidate the chemical structures and absolute configurations. The clerodane diterpenoids possessing a 6-OH group and no substitution at C-7 exhibited greater cytotoxic activity than others, with their IC50 values ranging from 0.3 to 2.9 μM. Isocaseamembrin E (7) exhibited antibacterial activity against Staphylococcus aureus, while isocaseamembrin E (7), corymbulosin X (8), caseargrewiin A (9), kurzipene A (10), and balanspene F (11) exhibited antibacterial activity against Bacillus cereus. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13659-024-00475-7.


Introduction
Casearia grewiifolia Vent.belongs to the Salicaceae family, previously placed in the defunct Flacourtiaceae family, and is widely distributed in the Indo-China to Queensland (Cook).In Thailand, C. grewiifolia, known as "Kruaipa", is a perennial evergreen tree (2-20 m high), growing widely in the north and northeast of Thailand.In Thai folk medicine, the four various parts of plant, root, leaves, seeds and flowers, have been traditionally utilized to treat liver dysfunction, skin diseases, hemorrhoids, as well as antidiarrheal and antipyretic agents, a tonic and febrifuge [1,2].Previous chemical studies of this plant revealed that the clerodane-type diterpenes were the main components of C. grewiifolia.Most clerodane diterpenes found in the genus Casearia are bicyclic diterpenoids with a cis-fused decalin ring system and a C-11-C-16 side chain at C-9 and a five-membered ring containing oxygen atom at C-18 and C-19 [2][3][4][5][6].These compounds have already been reported to exhibit cytotoxic, antimalarial, hypoglycemic, antiulcer, antiinflammatory, anti-snake venom, antiparasitic, and antimycobacterial activities [3][4][5][7][8][9].Recently, there has been reported that the grewiifolin C, a clerodane diterpene, downregulated PCSK9 and IDOL mRNA expression [6].In an attempt to search for structurally intriguing and bioactive clerodane diterpenes, a dichloromethane extract of C. grewiifolia has been investigated.In present work, we describe in detail of isolation and structure determination of eleven novel clerodane diterpenoids (1-4 and 12-18) along with nine known compounds (5-11, 19, and 20) and evaluation of their cytotoxicity, antibacterial activity, and aromatase inhibition.
Grewiifopene  13 C NMR data (Tables 1 and 3) of 3 was similar to those of 2, except for the different substituent at C-6; the appearance of an acetoxy group in 3 instead of a methoxy group in 2. This inference was implied by the downfield chemical shift of H-6 from δ H 3.30 (d, J = 10.3Hz) in 2 to 5.20 (m, overlapped) in 3, signals for 6-OAc (δ H 2.07/δ C 20.9) and ester carbonyl (δ C 170.1) as well as the HMBC experiments of H-19, H-7, and 6-OAc to C-6 (δ C 73.7) and H-6 to δ C 170.1 (Figure S26, Supporting Information).Inspection of 1 H-1 H coupling constants and NOESY spectra of 3 and 2 and their matched ECD curves (Fig. 5) conferred the absolute stereochemistry of 3 as shown.However, the coupling constants between H-6 and H-7 of 3 in CDCl 3 were difficult to measure directly due to signal overlap, but a spectrum obtained from a sample using benzene-d 6 as solvent (Table 1) provided the resolution of these two positions.Therefore, the chemical structure of 3 was purposed as shown.
Grewiifopene D (4) has an identical molecular formula to that of the co-occurring corymbulosin N (5).The resonance patterns of NMR data of 4 was identical to those of corymbulosin N (5) [10], excepted for the different stereochemistry at C-2 and C-19.However, the configuration at C-2 could be deduced by comparing the 13 C diagnostic shift of C-2 (δ c around 66.0 for 2R; δ c around 70.5 for 2S) [10,21].Therefore, the S-configuration at C-2 of 4 could be inferred from the 13 C chemical shift at δ C 70.5.The observed NOESY correlations of 19-OAc/6-OMe and H-19/H-11 (Fig. 3) implied the relative configurations of 19-OAc as β-oriented in 4. Based on these results, together with ECD comparison of its experimental with the calculated analysis (Fig. 4), its absolute stereochemistry was as shown.
Unfortunately, the stereochemistry at C-2′ of the 2-methybutyryl side chain in grewiifopenes A and D (1 and 4) could not be determined by comparing experimental ECD spectra with calculated ECD spectra method because its spectra of the 2′R and 2′S isomers of both compounds could not reveal the difference of diastereomers.These data are consistent with the previous reports for corymbulosin N (5) possessing the 2-methybutyryl side chain moiety [10].In an effort to further study, corymbulosin N (5) was used as authentic compounds because it was available in sufficient quantity by its conversion to the corresponding 2-methylbutyric acid benzyl ester using transesterification by heating in benzyl alcohol in the presence of a catalytic amount of dimethylaminopyridine (DMAP).In addition, the two authentic samples, S-and R/S-2-methylbutyric acid benzyl ester were synthesized from S-and R/S-2-methylbutyric acid, respectively.The ester products, including the desired product from transesterification of 5 and the two benzyl-2-methylbutyrate in S-and R/S-forms, were analyzed with the chiral-phase HPLC on CHIRALPAK ® AD-H column (40% CH 3 CN-H 2 O at flow rate 1 mL/min) revealing the S-configuration at C-2′ of 2-methylbutyryl side chain in corymbulosin N (5) (Fig. S118, Supporting Information).Based on the biogenesis rationale of compounds isolated from the same plant, the absolute configuration at C-2′ of 2-methylbutyryl side chain in grewiifopenes A and D (1 and 4), isocaseamembrin E (7), balanspene F (11), and grewiifopene I (16) should be established as S-configuration.
From the HRESIMS and NMR data of 12, its molecular formula was detected to be C 34 H 50 O 10 .The 1 H and 13 C NMR spectroscopic data (Tables 1 and 3) showed two sets of signals from the two isovaleryloxy groups.The comparison of the NMR data of compounds 2 and 12  indicated a structural difference by replacing the methoxy group at C-6 and an acetoxy group at C-7 in 2 with the hydroxy group and one of the isovaleryloxy groups in 12, respectively.This conclusion was in agreement with a series of the correlations of H-6/H-7/H-8/Me-17 in COSY experiment (Fig. S51, supporting information) and the characteristic correlations from H-7 to C-1′′ (δ C 173.9); H-10 and H-8 to C-6 (δ C 75.4); H-6 to C-19 (δ C 97.9); and Me-17 to C-7 (δ C 74.8), C-8 (δ C 41.8), and C-9 (δ C 39.1) in the HMBC experiment (Fig. S53, supporting information).The absorption in the IR spectrum at 3503 cm −1 implied the occurrence of the hydroxy moiety in 12. Based on the NOESY experiments (Fig. S54, supporting information) and 1 H-1 H coupling constants suggested that 12 had the same relative configurations as those of 2. Furthermore, the similarity of their ECD spectra of 12 and 2 (Fig. 5) indicated that the absolute stereochemistry of both 12 and 2 were virtually identical.Thus, compound 12 was elucidated and named as grewiifopene E.
Grewiifopene F (13) was colorless oil, and its molecular formula of C 37 H 48 O 10 was determined by HRESIMS analysis, which exhibited a [M+Na] + at m/z 675.3138 (calcd for C 37 H 48 NaO 10 , 675.3140).Comparison of the 1 H and 13 C NMR spectra (Tables 2 and 3) of 13 with 12 disclosed that compound 13 possessed a trans p-coumaroyl moiety at C-7 and a methoxy moiety at C-18 instead of the isovaleryloxy and acetoxy moieties in 12, respectively.The 1 H and 13 C NMR spectroscopic data (Tables 2  and 3) were consistent with the occurrence of a trans  had the same relative configurations as those of 12. Furthermore, ECD comparison of its experimental with the calculated analysis (Fig. 4), and its absolute stereochemistry was assigned as shown.
From the HRESIMS and NMR spectra of 14, its molecular formula was determined to be C 38 H 48 O 11 .Analyses of the 1 H and 13 C NMR data (Tables 1 and 3) of 14 were closely similar to those of 13, excepted that a methoxy group at C-18 was substituted with an acetoxy group in 14.This evidence was verified by the downfield shift of H-18 from δ H 5.49 (t, J = 1.5 Hz) in 13 to 6.74 (s) in 14.NOESY experiments (Fig. S74, supporting information) and 1 H-1 H coupling constants of 13 and 14 indicated the identical relative stereochemistry of 13 and 14, except for the H-18 position, which resonated as a singlet due to the disappearance of homoallylic 5 J coupling between H-2 and H-18 and no cross peak between H-18 and H-19 in the NOESY spectrum.This, in turn, would suggest the α-orientation for H-18; the other configuration of 14 was assigned to be the same as 13 based on the NOESY experiments and 1 H-1 H coupling constants.Consequently, the absolute stereochemistry of 14 was finally deduced as shown by the ECD calculation method (Fig. 4).Therefore, the structure of 14 (grewiifopene G) was assigned.
Grewiifopene I ( 16) was assigned the molecular formula C 29 H 42 O 8 , by HRESIMS at m/z 541.2782, [M+Na] + .The IR absorptions at 3456 cm −1 for the hydroxy and 1749, and 1729 cm −1 for ester carbonyl functionalities were detected.Analyses of the 1 H and 13 C NMR spectra (Tables 1 and 3) of 16 showed resonances similar to those of the co-occurring isocaseamembrin E (7) [11], except for the structural differences in the C-11-C-16 side chain moiety at C-9, which was identified as (Z)-3-methylpenta-1,3-diene.The relative stereochemistry of 16 was virtually similar to that of isocaseamembrin E (7) by analyzing their coupling constants in 1 H NMR and NOESY correlations (Fig. S94, supporting information).The comparison of the ECD curves of compound 16 with those of structural analogs 1, 2, 3, 12, and 15 (Fig. 5) penultimately assigned its absolute stereochemistry as shown.
Grewiifopene J (17) was isolated as a colorless oil.Its HRESIMS data gave a molecular formula of C 38 H 48 O 11 .Analyses of its 1 H and 13 C NMR spectra (Tables 2 and 3) of 17 displayed the same skeleton as 12, with the structural differences at C-9 and C-7.The NMR data exhibited resonances for (Z)-3-methylpenta-1,3-diene moiety at C-9 and trans p-coumaroyloxy group at C-7 in 17 instead of the 3-methylenepent-1-ene side chain and isovaleryloxy group, respectively, in 12. On the basis of the NOESY experiments (Fig. S104, supporting information) and 1 H-1 H coupling constants, compound 17 had the same relative stereochemistry as those of 12.The absolute stereochemistry of 17 was established as shown by using ECD comparison of its experimental with the calculated analysis.
Grewiifopene K (18) had the same molecular formula of C 38 H 48 O 11 as 17 based on a sodium ion adduct peak in the HRESIMS spectrum, indicating that 18 is isomeric with 17.The closely matched 1 H and 13 C NMR spectra (Tables 2 and 3) suggested that 18 possessed the same planar structure as 17, but they have differences in the geometry of the C-12/C-13 tri-substituted alkene.The NOESY cross peaks (Fig. 3) between H-12 and H-14; H-11 and Me-16 and the characteristic 13 C chemical shift of the olefinic methyl moiety (Me-16; δ C 12.1) indicated its E-configuration [21].Both the NOESY experiments (Fig. S114, supporting information) and 1 H-1 H coupling constants as well as ECD curves (Fig. 4) suggested that 18 had the same absolute configurations as those of 17.
In addition, the isolates were also evaluated for their antibacterial activity towards three strains including Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 6538) and Staphylococcus epidermidis (ATCC 12228) using penicillin G and gentamicin as the antibiotic controls (Table 5).However, due to insufficient materials for testing, only compounds 2, 3, 7-11, 15, 19, and 20 were subjected to evaluation.Most of the tested compounds, except for compound 7, did not show significant activity towards S. aureus and S. epidermidis.Compound 7 exhibited antibacterial activity against only S. aureus with the MIC and MBC values of 25 (50) µg/mL.Compounds 7-11 exhibited antibacterial activity toward B. cereus with MIC and MBC values of 6.25 (> 200), 25 (50), 100 (100), 12.5 (12.5), and 12.5 (25.0) μg/mL, respectively.Finally, the inhibitory activity of aromatase by using a CYP19 screening kit was investigated.The aromatase (also called estrogen synthase) is responsible for converting the non-aromatic ring A of androgens to estrogens, which are related to the proliferation of breast cancer cells.However, almost all the tested compounds (3, 5, 7, 9, 11, 12, 19, and 20) did not show activity at the concentration up to 12.15 µM (Table S11, supporting information).

Conclusions
In summary, a total of twenty isolates, including eleven novel clerodane diterpenoids, grewiifopenes A-K, as well as nine known compounds were isolated from the dichloromethane extract of C. grewiifolia.Their structures were determined by extensive spectroscopic analysis.Meanwhile, the absolute stereochemistry was established by comparing the experimental and calculated ECD data.A biological evaluation of some isolated compounds revealed that all tested clerodane diterpenoids possessed cytotoxic activities against a panel of cancer cell lines.On the other hand, only compound 7 exhibited antibacterial activity against S. aureus, while compounds 7-11 exhibited antibacterial activity against B. cereus.None of the tested compounds inhibited the aromatase.To the best of our knowledge, this is the first report on the antibacterial activity and inhibition of aromatase of clerodane diterpenoids from C. grewiifolia.

General experimental procedures
A JASCO P-1020 digital polarimeter measured at 590 nm (Na lamp D line) was used to measure the specific optical rotation values.A JASCO J-815 spectropolarimeter was utilized to provide the ECD spectra.A Perkin-Elmer Spectrum One spectrometer [attenuated total reflectance (ATR)] was used to obtain FT-IR spectra.A Bruker AVANCE 600 spectrometers was used to record 1D and 2D NMR spectra.A Bruker MicroTOFLC spectrometer was used to record the ESITOFMS (positive mode) data.The stationary phases for column chromatography (CC) are silica gel 60 (Merck, 70-200 mesh ASTM), Sephadex LH-20 gel (Ambersham Biosciences), and silica gel

Plant materials
The twigs and stems of Casearia grewiifolia Vent.(Salicaceae) were collected from the Sai Yok National Park, Kanchanaburi Province, Thailand (14.2495, 99.0270) in December 2020.One of the authors (T.T.) authenticated the plant, and a voucher specimen (SY16) has been deposited at the Forest Herbarium, Department of National Parks, Wildlife and Plant Conservation, Thailand.

Extraction and isolation
Air dried twigs and stems powder of C. grewiifolia (8.9 kg) were macerated two times with CH 2 Cl 2 at ambient temperature, and the resulting solvent was concentrated under vacuum to afford 50.0 g of the CH 2 Cl 2 extract.A 48 g of CH 2 Cl 2 extract was developed to column chromatography (CC) with silica gel with the mobile phase of hexane-acetone (a gradient from 100:0 to 0:100) and acetone-MeOH (a gradient from 100:0 to 0:100) provide sixteen fractions (A1-A16).The Sephadex LH-20 CC was then applied for fraction A6 (13.6 g) using CH 2 Cl 2 -MeOH in a ratio of 1:1 as eluent to give five fractions (B1-B5).Fraction B3 (2.7 g), was fractionated by silica gel CC with the mobile phase of hexane-EtOAc (a gradient from 100:0 to 0:100) providing seven fractions (C1-C7).A gradient elution of hexane and acetone was applied to silica gel CC for further separated fraction C5 (1.5 g) to provide eleven fractions (D1-D11).Using HPLC approach for purification of fraction D3 (69.0 mg) and eluting with a CH The corresponding S-2-methylbutyric acid benzyl ester (30 mg, 85%) was obtained by the same procedure using (S)-2-methylbutanoic acid.

Transesterification of compound 5
Corymbulosin N (5) (28.1 mg, 0.053 mmol), benzyl alcohol (0.5 mL, 4.8 mmol), and 4-dimethylaminopyridine (0.65 mg, 0.053 mmol) was heated to 120 °C in seal tube and stirred for 48 h.The reaction was allowed to cool to room temperature and then was purified with column chromatography with 5% EtOAc in hexane as eluent to remove a large amount of benzyl alcohol.The resulting was analyzed by chiral HPLC.

ECD computational methods
Spartan' 20 with MMFF94 molecular mechanics model (within 5 kcal/mol energy window) was used for a conformational search and Guassian 16 Rev.C.01 program was used for all DFT calculations [24,25].Further optimization of the obtained low-energy conformers was calculated using wB97XD/cc-PVDZ level of theory with IEFPCM of methanol solvent model.All optimized conformers were confirmed to be a true minimum of electronic potential energy by means of the vibrational frequency calculation at the same level without any detection of imaginary frequencies.Each conformer with the over 2% population was subjected to the ECD calculations, according to Boltzmann distribution law base on Gibbs free energies.Using TD-DFT calculations at the CAM-B3LYP/def2-SVP level of theory and the application of the IEFPCM of methanol solvent model [26] were computed the simulated ECD spectra of 1, 4, 8, and 15.
Thirty excited states of each conformer were calculated and the resulting ECD curves were developed using SpecDis with Boltzmann average all conformers and overlapping Gaussian function with an exponential halfwidth (σ = 0.35) [27,28].For the related enantiomers of 1, 4, 8, and 15, their theoretical ECD spectra derived from the direct inversion of their simulated ECD spectra.

Inoculum preparation
All tested species were grown on Mueller Hinton Agar (MHA) and incubated at 37 °C for 18-24 h.The bacterial colonies suspension in sterile normal saline was diluted until they reached a 1 × 10 6 CFC/mL concentration, which was then compared to the McFarland scale.This step provided a standard bacterial suspension (1 × 10 6 CFU/mL), which was used for the following assays.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The broth microdilution technique was used for the MIC and MBC determination, which was expressed according to Clinical and Laboratory Standards Institute (CLSI) standard M07 guideline [31].The compounds were prepared as a stock solution by dissolving in dimethyl sulfoxide (DMSO) with the concentration ranging from 0.195 to 200 µg/mL.DMSO and penicillin G or gentamicin were used as normal and standard drug control.The minimum inhibitory concentration (MIC) refers to the lowest antibacterial compound concentration at which no visual growth was observed, while the complete death of bacteria compared to the initial bacterial inoculum refers to the MBC (minimum bactericidal concentration).Three replicates were performed for each experiment.

Aromatase (CYP19) inhibition assay
The aromatase assay used a method previously designed by Stresser and co-workers [32].Letrozole, a positive control, exhibited an IC 50 value of 1.4 ± 0.3 nM for CYP19 inhibition.

Table
1H NMR spectroscopic data of compounds 1-4, 15, and 16 in CDCl 3 (600 MHz, J in Hz) a Recorded in C 6 D 6 b,c Overlapped signals

Table 4
Cytotoxicity of compounds 2, 3, and 5-20 a Inactive for cytotoxicity with < 50% inhibition at a concentration of 50 µg/mL b ND not determined d Etoposide and doxorubicin are used as positive controls Compound Cell lines (IC 50 , μM); values are expressed as mean ± s.d.(n = 3)

Table 5
Antimicrobial activity of compounds 2